Mutants of green fluorescent protein

ABSTRACT

The present invention provides mutants of the Green Fluorescent Protein (GFP) of  Aequorea victoria . Specifically provided by the present invention are nucleic acid molecules encoding mutant GFPs, the mutant GFPs encoded by these nucleic acid molecules, vectors and host cells comprising these nucleic acid molecules, and kits comprising one or more of the above as components. The invention also provides methods for producing these mutant GFPs. The fluorescence of these mutants is observable using fluorescein optics, making the mutant proteins of the present invention available for use in techniques such as fluorescence microscopy and flow cytometry using standard FITC filter sets. In addition, certain of these mutant proteins fluoresce when illuminated by white light, particularly when expressed at high levels in prokaryotic or eukaryotic host cells or when present in solution or in purified form at high concentrations. The mutant GFP sequences and peptides of the present invention are useful in the detection of transfection, in fluorescent labeling of proteins, in construction of fusion proteins allowing examination of intracellular protein expression, biochemistry and trafficking, and in other applications requiring the use of reporter genes.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/472,065, filed Dec. 23, 1999, which is a continuation ofU.S. patent application Ser. No. 08/970,762, filed Nov. 14, 1997 (nowabandoned), which claims the benefit of U.S. Provisional Application No.60/030,935, filed Nov. 15, 1996, the disclosures of which applicationsare entirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is in the fields of molecular and cellularbiology. More particularly, the invention is directed to mutants of thegenes encoding Green Fluorescent Protein (GFP) and the proteins encodedby these mutants. The mutant GFPs are used to allow detection ofeukaryotic and prokaryotic cells transfected or transformed withextrinsic genes, and to label proteins of interest to facilitate theirlocalization within viable cells.

[0004] 2. Related Art

[0005] Transfection of Foreign Genes

[0006] To study the function of a gene, a technique that is commonlyemployed is the transfer of the gene into a new cellular environment.This process, called “transfection,” provides several advantages to thegenetic scientist. For example, the cellular protein encoded by the genecan often be more easily studied by transferring the gene into a cell ororganism that normally does not produce the protein, and then examiningthe effect of this protein on the host cell. The existence and functionof regulatory genetic sequences (e.g., promoters, inhibitors andenhancers) may be elucidated by transfection of foreign genes into cellscontaining the regulatory sequences. The transfer of non-native oraltered genes into a host cell also allows for large-scale production ofthe proteins encoded by the genes, a process upon which much of thecurrent biotechnology industry is based. Transfection of plant embryoswith foreign genes has provided genetically engineered plants that aremore resistant to adverse environmental conditions or that are morenutritionally rich. Finally, gene transfer methods allow theintroduction of new or mutated genes into whole organisms. This lattercapability provides the opportunity for the construction of stablemodels of mammalian diseases, for large-scale production of proteins inthe milk of transgenic lactating animals, and for the possibility ofgenetic therapy for certain diseases.

[0007] A variety of techniques has been used to transfect non-nativegenes into cells (reviewed in Sambrook, J., et al., Molecular Cloning, aLaboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 16.30-16.55 (1989); Watson, J. D., et al.,Recombinant DNA, 2nd Ed., New York: W. H. Freeman and Co., pp. 213-234(1992)). These techniques include biological methods such as the use ofviruses (e.g., adenovirus or certain retroviruses for mammalian cells,baculovirus for insect cells and bacteriophages for bacterial cells) orbacteria (e.g., Agrobacterium for plant cells), chemical methods such ascalcium phosphate precipitation, DEAE-dextran-mediated endocytosis orliposome-mediated transfection, and physical methods such aselectroporation or direct microinjection. For transfection of mammaliancells, the techniques most commonly employed currently arevirus-mediated transfection, lipofection and electroporation.

[0008] Detection of Gene Transfer

[0009] Regardless of the method used, however, simply attempting totransfect a cell does not guarantee that a majority (or even any) of thetarget cells will take up and/or express the exogenous DNA. Indeed, ithas been suggested that the success rate of even the most optimaltechniques used for transfection results in stable transfer of exogenousDNA is far less than 1% (Watson, J. D., et al, Recombinant DNA, 2nd Ed.,New York: W. H. Freeman and Co., pp. 216, 218 (1992)). Thus, it isusually critical to determine which target cells have received and/orincorporated the gene(s) being transfected, for which a number ofmethodologies have been used.

[0010] Expression

[0011] The most obvious of these methods is to simply examine the targetcells for expression of the exogenous gene. In this method, thetransfected cells are grown in vitro and assayed for the presence of theprotein encoded by the transferred gene. These assays are usuallyaccomplished using immunological techniques such as Western blotting,ELISA or RIA. This type of technique is only useful, however, if theprotein is produced in relatively high amounts (generally at themicrogram level or above) and if suitable antibodies are available,neither of which is the case for some transfected genes.

[0012] In those cases where protein expression cannot be examined,incorporation of exogenous genes can be determined by assaying thetarget cells for production of the mRNAs corresponding to thetransferred genes. One very common technique for this determination isNorthern blotting (Alwine, J. C., et al., Proc. Natl. Acad. Sci. USA74:5350-5354, 1977), in which RNA molecules are isolated from cells,separated by gel electrophoresis and electroblotted onto a solid support(e.g., nitrocellulose or nylon). The solid support is then overlaid withradiolabelled cDNAs corresponding to the transfected gene, whichhybridize on the solid support to their complementary mRNAs. Afterexposing the blot to photographic film, the samples containing theexpressed transgene are easily determined. While this method is moresensitive than those directly measuring protein expression, Northernblotting still relies on actual expression of the gene by the targetcells, which is not always the case.

[0013] Selection

[0014] Another method for determining gene transfer, alternative todirectly measuring gene expression, is to examine the effect of the geneon the transfected cells. For example, some transfected genes willconfer upon their host cells the ability to grow in selective culturemedia or under some other environmental stress which non-transfectedcells cannot tolerate. Genes of interest are often engineered intosequences conferring, for example, antibiotic resistance upon therecipient cells. Transfectants with these constructs will thus carry notonly the gene of interest but also the antibiotic resistance gene whichallows them to grow in antibiotic-containing media. Sincenon-transfected cells will not possess this resistance, any cell able togrow in media containing antibiotic will contain the resistance marker(the so-called “selectable marker”) and the transgene that is linked toit. Selectable markers commonly used in such an approach are theneomycin (neo), ampicillin (amp) and hygromycin (hyg) resistance genes.

[0015] In the same way, selectable markers conferring on the transfectedcells a metabolic advantage (e.g., ability to grow in nutrient-deficientmedia) have been used successfully. Examples of these types ofselectable markers include thymidine kinase (Bacchetti, S., and Graham,F. L., Proc. Natl. Acad. Sci. USA 74:1590-1594 (1977); Wigler, M., etal., Cell 11:223-232 (1977)) and xanthine-guaninephosphoribosyltransferase (Mulligan, R. C., and Berg, P., Proc. Natl.Acad. Sci. USA 78:2072-2076 (1981)), which impart to their recipientsthe ability to grow, using metabolic rescue pathways encoded by themarker genes, in media that inhibit vital metabolic pathways innon-transfected cells. Again, any cells able to grow in such media willcontain the transgene linked to the marker gene.

[0016] Selection methods such as these often require weeks of culturingof the cells, continuously under selective pressure, to provide arelatively pure population of stable transfectants. Many uses oftransfected cells, however, are conducted within hours of transfection,far too soon to determine transfection success using either theexpression or selection methods described above. These types ofapplications are facilitated by a third approach—the use of “reportergenes”.

[0017] Reporter Genes

[0018] Reporter genes are analogous to selectable markers in that theyare co-transfected into recipient cells with the gene of interest, andprovide a means by which transfection success may be determined. Unlikeselectable markers, however, reporter genes typically do not confer anyparticular advantage to the recipient cell. Instead reporter genes, astheir name implies, indicate to the observer (via some phenotypicactivity) which cells have incorporated the reporter gene and thus thegene of interest to which it is linked. A number of reporter genes havebeen used, including those operating by biochemical or fluorescentmechanisms, each with its own advantages and limitations.

[0019] Biochemical Reporter Genes

[0020] Some commonly used reporter genes encode enzymes or otherbiochemical markers which, when active in the transfected cells, causesome visible change in the cells or their environment upon addition ofthe appropriate substrate. Two examples of this type of reportersequence are the E. coli genes lacZ (encoding β-galactosidase or“β-gal”) and gusA or iudA (encoding β-glucuronidase or “β-glu”); theformer is often used as a reporter gene in animal cells (Hall, C. V., etal, J. Mol. Appl. Genet 2:101-109 (1983); Cui, C., et al., TrangenicRes. 3:182-194 (1994)), the latter in plant cells (Jefferson, R. A.,Nature 342:837-838 (1989); Watson, J. D., et al., Recombinant DNA, 2ndEd., New York: W. H. Freeman and Co., pp. 281-282 (1992); Hull, G. A.,and Devic, M., Meth. Mol. Biol. 49:125-141 (1995)). These bacterialsequences are useful as reporter genes because the recipient cells,prior to transfection, express extremely low levels (if any) of theenzyme encoded by the reporter gene. When transfected cells expressingthe reporter gene are incubated with an appropriate substrate (e.g.,X-gal for β-gal or X-gluc for β-glu), a colored or fluorescent productis formed which can be detected and quantitated histochemically orfluorimetrically.

[0021] Another often-used reporter gene is the bacterial gene encodingchloramphenicol acetyltransferase (CAT), which catalyzes the addition ofacetyl groups to the antibiotic chloramphenicol (Gorman, C. M., et al.,Mol. Cell. Biol. 2:1044-1051 (1982); Neumann, J. R., et al.,BioTechniques 5:444-446 (1987); Eastman, A., BioTechniques 5:730-732(1987); Felgner, P. L., et al., Ann. N.Y. Acad. Sci. 772:126-139(1995)). After transfection, recipient cells are lysed and the lysatesare incubated with radiolabelled chloramphenicol and an acetyl donorsuch as acetyl-CoA, or with unlabeled chloramphenicol and radiolabeledacetyl-CoA (Sleigh, M. J., Anal. Biochem. 156:251-256 (1986)). Ifexpressed in the cells, CAT transfers acetyl groups to chloramphenicol,which is then easily assayed by chromatographic techniques, therebygiving an indication of the incorporation of the co-transfected gene ofinterest by the recipient cells.

[0022] Using reporter genes in this way, populations of cells, or evensingle cells, can be rapidly assayed for their incorporation of theexogenous gene linked to the reporter gene. Since they do not relydirectly on the expression of the gene of interest, assays oftransfection success using reporter genes are usually simpler and moresensitive than those measuring mRNA or protein production from thetransgene (Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W.H. Freeman and Co., p. 155 (1992)). However, the use of reporter genesis severely limited in that it usually requires sacrifice (fixation) ofthe cells prior to assay, and therefore cannot be used for assayingliving cells or cultures. Thus, alternative means for determining theincorporation of the transgene in viable cells have been developed.

[0023] Fluorescent Reporter Genes

[0024] An example of viable reporter genes that are rapidly gainingwidespread use are those that are fluorescence-based. These genes encodeproteins which are either naturally fluorescent or which convert asubstrate from nonfluorescent to fluorescent. Assays using this type ofreporter gene are non-destructive and, owing to the availability ofsophisticated fluorescence detection systems, are often more sensitivethan biochemical reporter gene assays.

[0025] One example of a fluorescence reporter gene is theluciferin-luciferase system (Bronstein, I., et al., Anal. Biochem.219:169-181 (1994)). This system utilizes the gene for luciferase, anATPase enzyme isolated from fireflies (Gould, S. J., and Subramani, S.,Anal. Biochem. 175:5-13 (1988)) and other beetles (Wood, K. V., et al.,J. Biolumin. Chemilumin. 4:289-301 (1989)), or from certainbioluminescent bacteria (Stewart, G. S., and Williams, P., J. Gen.Microbial 138:1289-1300 (1992); Langridge, W., et al., J. Biolumin.Chemilumin. 9:185-200 (1994)). For use as a reporter gene, theluciferase gene is placed into a vector also containing the gene ofinterest, or separate vectors containing the luciferase gene and thegene of interest are mixed together. Cells are then transfected with thevector(s) and treated with the luciferase substrate luciferin which isrendered luminescent (and impermeant) intracellularly by the action ofthe luciferase. Cells containing the luciferase gene, and thus the geneof interest linked to it, can then be rapidly and sensitively observedusing luminescence detectors such as luminometers.

[0026] To provide a further increase in sensitivity, attempts have beenmade to use genes from certain cyanobacteria which encode naturallyfluorescent phycobiliproteins such as phycoerythrin and phycocyanin.These proteins are among the most highly fluorescent known (Oi, V. T.,et al., J. Cell Biol. 93:981-986 (1982)), and systems have beendeveloped that are able to detect the fluorescence emitted from aslittle as one phycobiliprotein molecule (Peck, K., et al., Proc. Natl.Acad. Sci. USA 86:4087-4091 (1989)). Phycobiliproteins also have theadvantage of being naturally fluorescent, thus eliminating thetime-consuming steps of the addition of exogenous substrates for theirdetection as is required for luciferase and biochemical reporter genes.However, the phycobiliproteins have proven extremely difficult toengineer into gene constructs in such a way as to maintain theirfluorescence (Heim, R., et al., Proc. Natl. Acad. Sci. USA91:12501-12504 (1994)), and thus are not commonly used as reporter genesin assaying the transfection of mammalian cells.

[0027] Thus, the ideal reporter gene would encode a naturallyfluorescent protein (for ease of use following transfection) that ishighly fluorescent (for increased sensitivity) and easily engineered(for maintenance of fluorescence). Such a system has recently beendeveloped, using the Green Fluorescent Proteins (GFPs) isolated fromcertain marine cnidarians.

[0028] GFP

[0029] Overview

[0030] GFPs are involved in bioluminescence in a variety of marineinvertebrates, including jellyfish such as Aequorea spp. (Morise, H., etal., Biochemistry 13:2656-2662 (1974); Prendergast, F. G., and Mann, K.G., Biochemistry 17:3448-3453 (1978); Ward, W. W., Photochem. Photobiol.Rev. 4:1-57 (1979) and the sea pansy Renilla reniformis (Ward, W. W.,and Cormier, M. J., Photochem. Photobiol. 27:389-396 (1978); Ward, W.W., et al., Photochem. Photobiol. 31:611-615 (1980)). The GFP isolatedfrom Aequorea victoria has been cloned and the primary amino acidstructure has been deduced (FIG. 2; Prasher, D. C., et al., Gene111:229-233 (1992)) (SEQ ID NOs:3, 4). The chromophore of A. victoriaGFP is a hexapeptide composed of amino acid residues 64-69 in which theamino acids at positions 65-67 (serine, tyrosine and glycine) form aheterocyclic ring (Prasher, D. C., et al., Gene 111:229-233 (1992);Cody, C. W., et al., Biochemistry 32:1212-1218 (1993)). Resolution ofthe crystal structure of GFP has shown that the chromophore is containedin a central α-helical region surrounded by an 11-stranded β-barrel(Ormö, M., et al., Science 273:1392-1395 (1996); Yang, F., et al.,Nature Biotech. 14:1246-1251 (1996)). Upon purification, native GFPdemonstrates an absorption maximum at 395 nanometers (nm) and anemission maximum at 509 nm (Morise, H., et al., Biochemistry13:2656-2662 (1974);Ward, W. W., et al., Photochem. Photobiol.31:611-615 (1980)) with exceptionally stable and virtuallynon-photobleaching fluorescence (Chalfie, M., et al., Science263:802-805 (1994)).

[0031] While GFP has been used as a fluorescent label in proteinlocalization and conformation studies (Heim, R., et al., Proc. Natl.Acad. Sci. USA 91:1250-1254 (1994); Yokoe, H., and Meyer, T., NatureBiotech. 14:1252-1256 (1996)), it has gained increased attention in thefield of molecular genetics since the demonstration of its utility as areporter gene in transfected prokaryotic and eukaryotic cells (Chalfie,M., et al., Science 263:802-805 (1994); Heim, R., et al., Proc. Natl.Acad. Sci. USA 91:1250-1254 (1994); Wang, S., and Hazelrigg, T., Nature369:400-403 (1994)). GFP has also been used in fluorescence resonanceenergy transfer studies of protein-protein interactions (Heim, R., andTsien, R. Y., Curr. Biol. 6:178-182 (1996)). Since GFP is naturallyfluorescent, exogenous substrates and cofactors are not necessary forinduction of fluorescence, thus providing GFP an advantage over thebiochemical, luminescent and other fluorescent reporter genes describedabove. Visualization of GFP fluorescence does not require the fixationsteps necessary with biochemical reporters such as β-gal and β-glu, nordoes it require extraction from the cell prior to assay as may berequired with luciferase; thus, GFP is suitable for use in proceduresrequiring continued viability of transfected cells. In addition, sincethe GFP cDNA containing the complete coding region is less than 1kilobase in size (Prasher, D. C., et al, Gene 111:229-233 (1992)), it iseasily manipulated and inserted into a variety of vectors for use increating stable transfectants (Chalfie, M., et al., Science 263:802-805(1994)).

[0032] Despite these advantages, however, the use of wildtype GFP has afew limitations. For example, the excitation and emission maxima ofwildtype GFP are not within the range of wavelengths of standardfluorescence optics (at which GFP demonstrates relatively low quantumyield (i.e., low intensity of fluorescence)). In addition, GFP shows lowefficiency of transcription in mammalian cells upon transfection and ispackaged into low-solubility inclusion bodies in bacteria (thusproviding difficulty in purification). These limitations have beenovercome to a limited extent via the introduction of selected pointmutations into the sequence of wildtype GFP.

[0033] GFP Mutants

[0034] One of the earliest mutation studies of GFP, in which thetyrosine residue at position 66 in the wildtype protein (“wt-GFP”) wasreplaced with a histidine residue, resulted in a mutant protein whichfluoresced blue instead of green when excited with ultraviolet (UV)light (Heim, R., et al, Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)).This mutant protein not only provided a capacity for two distinguishablewavelengths for use in studies comparing independent proteins and geneexpression events, but also demonstrated that single point mutations inGFP could induce drastic changes in the photochemistry of the protein.Three other sets of specific point mutations have been shown to increasethe excitation and emission maxima of GFP such that they fall wellwithin the range of standard fluorescein optics (Ehrig, T., et al., FEBSLetts. 367:163-166 (1995); Delagrave, S., et al., Bio/Technology13:151-154 (1995); Heim, R., and Tsien, R., Curr. Biol. 6:178-182(1996)), thus permitting the use of GFP with standard laboratoryfluorescence detection systems. The problem of low quantum yield bywt-GFP has been partially addressed by mutating the serine residue atposition 65 to a threonine (“S65T”), either without (Heim, R., et al,Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)) or with (Cormack, B., etal., Gene 173:33-38 (1996)) a concomitant mutation at position 64, or bymutating other residues in the non-chromophore region (Crameri, A., etal., Nature Biotech. 14:315-319 (1996)). The S65T mutation also appearsto improve the rate of fluorophore formation in transfected cells byapproximately four-fold over wt-GFP, thus allowing earlier and moresensitive detection of transfection with this mutant than with wt-GFP(Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)). Bycombining the S65T mutation with a mutation at position 64 replacingphenylalanine with leucine, approximately 90% of the mutant GFPexpressed in bacteria is soluble, thus improving protein purificationand yields (Cormack, B., et al., Gene 173:33-38 (1996)). Another seriesof mutations results in a mutant fusion GFP consisting of linked blue-and green-fluorescing proteins which have proven useful in studies ofprotein localization, targeting and processing (Heim, R., and Tsien, R.Y., Curr. Biol. 6:178-182 (1996)). Analogously, chimeric constructscomprising GFP linked to other proteins have been used in studies of ionchannel expression and function (Marshall, J., et al., Neuron 14:211-215(1995)), and in organelle targeting studies where they have provided ameans for selectively and distinctively labeling the organelles ofliving cells (Rizzuto et al., Curr. Biol. 6:183-188 (1996)). Finally, bycombining the S65T mutation with other mutations throughout thenonchromophore regions of the wt-GFP gene, a “humanized” mutant GFP (SEQID NOs:1, 2) has been produced that not only shows a significantincrease in fluorescence intensity and rate of fluorophore formationover wt-GFP (via the S65T mutation) but also demonstrates a 22-foldincreased expression efficiency in mammalian cells (Evans, K., et al.,FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol 70:4646-4654(1996)). This humanization was achieved via 92 base substitutions (in 88codons) to the wt-GFP gene which were amino acid-conservative and whichwere made to provide a pattern of codon usage more closely resemblingthat of mammalian cells, as opposed to the jellyfish codon patternsfound in the wt-GFP gene which are less efficiently translated inmammalian cells. A summary of these GFP chromophore mutants is presentedin Table 1. TABLE 1 GFP Chromophore Mutants. Amino Acid Residue Number:Mutant 64 65 66 Reference¹ (Wildtype) Phe Ser Tyr Prasher et al., 1992GreenLantern-1 Phe Thr Tyr Evans et al., 1996 Humanized GFP Phe Thr TyrZolotukhin et al., 1996 Y66H Phe Ser His Heim et al., 1994 Y66W Phe SerTrp Y66F Phe Ser Phe RSGFP1 Gly Ser Tyr Delagrave et al., 1995 RSGFP2Leu Leu Tyr RSGFP3 Gly Cys Tyr RSGFP4 Met Gly Tyr RSGFP6 Val Ala TyrRSGFP7 Leu Cys Tyr S65A Phe Ala Tyr Heim et al., 1996 S65L Phe Leu TyrS65C Phe Cys Tyr S65T Phe Thr Tyr GFPmut1 Leu Thr Tyr Cormack et al.,1996

[0035] Despite some success in overcoming certain of the above-describedlimitations of GFPs, the sensitivity of GFP as a reporter gene (measuredas percentage of positive cells) is not as high as that of standardbiochemical reporter genes such as β-gal (Evans, K., et al., FOCUS18(2):40-43 (1996)). In addition, the use of GFP as a reporter gene or aprotein tag requires the use of fluorescent excitation and emissionoptics, which increases user expense and which is more technicallychallenging than the use of visible or white light optics often usedwith standard reporters such as β-gal. Thus, a need currently exists foradditional GFP variants which are more highly fluorescent, humanized,rapidly expressed in mammalian cells, capable of being observed usingstandard white light optics, and which provide an increased level ofsensitivity.

SUMMARY OF THE INVENTION

[0036] It is thus an object of the present invention to provide mutantGFP cDNAs and proteins. In one aspect, the invention relates to suchmutant GFP cDNAs which, when transfected into prokaryotic (e.g.,bacterial) or eukaryotic (e.g., mammalian) cells, increase thesensitivity of detection (measured as percentage or number of positivecells). The present invention thus provides nucleic acid moleculesencoding mutant GFPs, wherein the mutant GFPs have an amino acidsequence comprising an amino acid residue lacking an aromatic ringstructure at position 64 and an amino acid residue having a side chainno longer than two carbon atoms in length at position 65. Preferably,(a) if the residue at position 64 is leucine then the residue atposition 65 is not cysteine or threonine; (b) if the residue at position64 is valine then the residue at position 65 is not alanine; (c) if theresidue at position 64 is methionine then the residue at position 65 isnot glycine; and (d) if the residue at position 64 is glycine then theresidue at position 65 is not cysteine. The invention is particularlydirected to such nucleic acid molecules encoding mutant GFPs wherein theamino acid residue at position 64 is alanine, valine, leucine,isoleucine, proline, methionine, glycine, serine, threonine, cysteine,alanine, asparagine, glutamine, aspartic acid or glutamic acid, mostpreferably cysteine or methionine. The invention is also particularlydirected to such nucleic acid molecules encoding mutant GFPs wherein theamino acid residue at position 65 is alanine, glycine, threonine,cysteine, asparagine or aspartic acid, most preferably alanine. Inparticular, the invention provides nucleic acid molecules encodingmutant GFPs wherein the amino acid at position 64 is cysteine ormethionine and the amino acid at position 65 is alanine, and nucleicacid molecules encoding mutant GFPs having an amino acid sequence as setforth in either SEQ ID NO:5 or SEQ ID NO:6.

[0037] In additional aspects, the invention provides mutant GFPs encodedby any of the above-described nucleic acid molecules, vectors(particularly expression vectors) comprising these nucleic acidmolecules, host cells (prokaryotic or eukaryotic (including mammalian))comprising these nucleic acid molecules or vectors, and compositionscomprising plasmid pGreenLantern-2/A1 or plasmid pGreenLantern-2/A4. Theinvention also provides methods for producing a mutant GFP, comprisingculturing the above-described host cells under conditions favoring theproduction of a mutant GFP and isolating the mutant GFP from the hostcell. The invention also provides mutant GFPs produced by these methods,particularly wherein the mutant GFPs emit fluorescent light whenilluminated with white light. The invention also relates to compositionscomprising the above-described mutant GFPs.

[0038] The invention is further directed to kits for transfecting a hostcell with the nucleic acid molecules encoding the present mutant GFPs,such kits comprising at least one container containing a nucleic acidmolecule encoding a mutant GFP such as those described above, whichpreferably comprises plasmid pGreenLantern-2/A1 or plasmidpGreenLantern-2/A4. These kits of the invention may optionally furthercomprise at least one additional container containing a reagent,preferably comprising a liposome and most preferably LIPOFECTAMINE™, fordelivering a mutant GFP nucleic acid molecule into a host cell.

[0039] The invention is further directed to kits for labeling apolypeptide with the present mutant GFPs, such kits comprising at leastone container containing a mutant GFP such as those described above,preferably a mutant GFP having an amino acid sequence as set forth inSEQ ID NO:5 or SEQ ID NO:6. These kits of the invention may optionallyfurther comprise at least one additional container containing a reagentfor covalently linking this mutant GFP to the target polypeptide.

[0040] The fluorescence of all of the GFP mutants provided by thepresent invention is observable with fluorescein optics, making thesemutant proteins amenable to use in techniques such as fluorescencemicroscopy and flow cytometry using standard FITC filter sets. Inaddition, the fluorescence of certain of the present GFP mutants,particularly those having amino acid sequences as set forth in SEQ IDNOs: 5 and 6, is visible using standard white light optics (e.g.,incandescent or fluorescent indoor lighting, or sunlight). The nucleicacid molecules and mutant GFPs provided by the present invention thuscontribute improved tools for detection of transfection, for fluorescentlabeling of proteins, for construction of fusion proteins allowingexamination of intracellular protein expression, biochemistry andtrafficking, and for other applications requiring the use of reportergenes.

[0041] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of the following drawings anddescription of the invention, and of the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0042]FIG. 1 is a depiction of the nucleotide (SEQ ID NO:1) and deducedamino acid (SEQ ID NO:2) sequences of humanized S65T mutant A. victoriaGreen Fluorescent Protein cDNA (after Zolotukhin, S., et al., J. Virol.70:4646-4654 (1996)).

[0043]FIG. 2 is a depiction of the nucleotide (SEQ ID NO:3) and deducedamino acid (SEQ ID NO:4) sequences of A. victoria Green FluorescentProtein cDNA (after Prasher, D. C., et al., Gene 111:229-233 (1992)).

[0044]FIG. 3 is a depiction of the amino acid sequence (SEQ ID NO:5) ofthe A1 GFP mutant.

[0045]FIG. 4 is a depiction of the amino acid sequence (SEQ ID NO:6) ofthe A4 GFP mutant.

[0046]FIG. 5 is a structural map of plasmid pGreenLante-1.

[0047]FIG. 6 is a structural map of plasmid pGreenLantern-2.

[0048]FIG. 7 is a fluorescence photomicrograph of CHO-K1 cells viewed 24hours after transfection with the A1 GFP mutant (plasmidpGreenLantern-2/A1).

[0049]FIG. 8 is a fluorescence photomicrograph of CHO-K1 cells viewed 24hours after transfection with the A4 GFP mutant (plasmidpGreenLantern-2/A4).

[0050]FIG. 9 is a fluorescence photomicrograph of negative controlCHO-K1 cells viewed 24 hours after transfection with the pGreenLantern-2backbone.

[0051]FIG. 10 is a bar graph demonstrating the fluorescence of CHO-K1cells determined by flow cytometry 24 hours after transfection withvarious GFP mutants.

[0052]FIG. 11 is a bar graph demonstrating the fluorescence of CHO-K1cells determined by flow cytometry 48 hours after transfection withvarious GFP mutants.

[0053]FIG. 12 is a structural map of plasmid pProEX HTb.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Overview

[0055] The present invention provides nucleic acid molecules encodingmutant GFPs, vectors and host cells comprising these nucleic acidmolecules, the mutant GFP polypeptides, and methods for producing mutantGFPs. Although specific plasmids, vectors, promoters, selection methodsand host cells are disclosed and used herein and in the Examples, otherpromoters, vectors, selection methods and host cells, both prokaryoticand eukaryotic, are well-known to one of ordinary skill in the art andmay be used to practice the present invention without departing from thescope of the invention or any of the embodiments thereof.

[0056] In the present invention, GFPs with selective point mutations atamino acid positions 64 and 65 have been constructed and analyzed. Ingeneral, it has been discovered in the present invention that when theamino acid residue at position 64 (phenylalanine in wt-GFP) is mutatedto an amino acid lacking an aromatic ring (e.g., alanine, valine,leucine, isoleucine, proline, methionine, glycine, serine, threonine,cysteine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine or histidine), an increase in fluorescence quantum yield isobserved. Increased fluorescence intensity is also observed when theamino acid residue at position 65 (serine in wt-GFP) is mutated to anamino acid having a side chain consisting of no more than two carbonatoms (e.g., alanine, glycine, threonine, cysteine, asparagine oraspartic acid), which induce a significant “red-shift” in excitationmaximum from ultraviolet to visible blue wavelengths and a singleexcitation maximum instead of a dual excitation maximum as in thewildtype protein. Together, these general results indicate that in orderto construct GFP mutants with a dramatic increase in fluorescenceintensity from wt-GFP, either position 64 or position 65 should containa reactive amino acid, although particular amino acids appear to bepreferred at each position as described below. Furthermore, it has beenunexpectedly discovered that several of the mutant GFPs of the presentinvention, unlike those previously known in the art, will emitfluorescence when illuminated by white light (e.g., incandescent orfluorescent indoor lighting, or sunlight).

[0057] Accordingly, in the present invention, specific mutations areintroduced into positions 64 and 65 of the wt-GFP cDNA sequence (SEQ IDNO:3). Alternatively, increased expression of the present mutant GFPsmay be obtained by introducing the preferred mutations into a humanizedGFP gene such as that described previously (SEQ ID NO:1) (Evans, K., etal., FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J Virol.70:4646-4654 (1996)).

[0058] Construction of GFP Mutants

[0059] Preparation of GFP Plasmids

[0060] The wt-GFP may be cloned from its natural source, Aequoreavictoria, as described (Prasher, D. C., et al., Gene 111:229-233(1992)). More preferably, GFP cDNA to be mutated is contained within aplasmid construct or vector, preferably an expression vector, suitablefor use in transfecting mammalian cells, such as pRAY-1 wherein thewt-GFP cDNA is under the control of the human cytomegalovirus (CMV)enhancer/promoter (Marshall, J., et al., Neuron 14:211-215 (1995)). Mostpreferably, to provide for optimum expression of the mutant GFPs inmammalian cells, the humanized S65T mutant GFP cDNA (Evans, K., et al.,FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654(1996)) under control of the CMV enhancer/promoter may be used,contained in plasmid pGreenLantern-1 (FIG. 5), which is availablecommercially from Invitrogen Corporation, Carlsbad, Calif.

[0061] The above-described plasmids may be used directly for preparationof mutant GFP cDNAs according to the present invention. Alternatively, astop codon in the 5′ multiple cloning site of pGreenLantern-1 may beshifted out of frame by oligonucleotide ligation methods to allow themutant GFPs of the present invention to be used in the construction offusions between GFP and other proteins, as described below.

[0062] Mutations to GFP cDNA

[0063] A variety of random or site-directed mutagenic techniques may beused to prepare the mutant GFPs of the present invention. Appropriatemethods include chemical mutagenesis using, for example, sodiumbisulfite or hydroxylamine (Myers, R. M., et al., Science 229:242-247(1985); Sikorski, R. S., and Boeke, J. D., Meth. Enzymol. 194:302-318(1991)), linker insertion mutagenesis (Heffron, F., et al., Proc. Natl.Acad. Sci. USA 75:6012-6016 (1978)), deletion mutagenesis (Lai, C. J.,and Nathans, D., J. Mol. Biol. 89:179-193 (1974); McKnight, S. L., andKingsbury, R., Science 217:316-324 (1982)), enzyme misincorporationmutagenesis (Shortle, D., et al., Proc. Natl. Acad. Sci. USA79:1588-1592 (1982)), oligonucleotide-directed mutagenesis (Hutchinson,C A., et al., J. Biol. Chem. 253:6551-6560 (1978); Zoller, M. J., andSmith, M., Nucl. Acids Res. 10:6487-6500 (1982); Taylor, J. W., et al.,Nucl. Acids Res. 13:8765-8785 (1985)), and cassette mutagenesis (Lo,K.-M., et al., Proc. Natl. Acad. Sci. USA 81:2285-2289 (1984); Wells, J.A., et al., Gene 34:315-323 (1985)). To improve the fidelity andefficiency of mutagenesis, the use of the polymerase chain reaction(PCR) in accomplishing GFP mutagenesis by one or more of the foregoingmethods is preferred (Higuchi, R., et al., Nucl. Acids Res. 16:7351-7367(1988); Leung, D. W., et al., Technique 1:11-15 (1989); Clackson, T.,and Winter, G., Nucl. Acids Res. 17:10163-10170 (1989)).

[0064] Most preferably, mutations are made to GFP cDNA by uracil DNAglycosylase (UDG) mutagenesis using PCR amplification (Nisson, P., etal., PCR Meth. Appl. 1:120-123 (1991)). In this approach, the plasmidcontaining GFP cDNA, most preferably pGreenLantern-1 comprisinghumanized S65T GFP (FIG. 5), is used as the PCR template, and a sense orantisense primer consisting essentially of an oligonucleotide containingat least one mismatched nucleotide (available commercially fromInvitrogen Corporation, Carlsbad, Calif.) is added to the reactionmixture. Amplification reaction mixtures most preferably contain 1×PCRbuffer, about 10 micromolar each of deoxyATP, deoxyTTP, deoxyCTP anddeoxyGTP, about 25 picomoles each of sense and antisense primers andabout 10 nanograms of template. PCR is performed by techniques that areroutine in the art, and after at least five PCR cycles, samples of thereaction mixture are treated with UDG, most preferably for 30 minutes at37° C., as described (Nisson, P., et al., PCR Meth. Appl. 1:120-123(1991)).

[0065] The mutated GFP nucleic acid molecules preferably will comprisenucleic acid sequences encoding mutant proteins in which one or moreamino acid residues have been mutated from the wildtype amino acidsequence set forth in FIG. 2 and SEQ ID NO:4. Such mutations mayinclude, for example, substitutions, deletions, insertions ormodifications, and preferably are amino acid substitutions. Particularlypreferred are amino acid substitutions occurring in the three amino acidchromophore of GFP at residues 64, 65 and 66 of the wildtype GFPsequence (FIG. 2 and SEQ ID NO:4), wherein the phenylalanine residue atposition 64 (Phe64), the serine residue at position 65 (Ser65), and thetyrosine residue at position 66 (Tyr66), are each individually, or alltogether, replaced by other amino acid residues. More preferred mutantGFPs of the invention include, but are not limited to, those with thefollowing substitutions from the wildtype GFP sequence shown in FIG. 2and SEQ ID NO:4:

[0066] serine 65 replaced by threonine (Ser65→Thr);

[0067] Phe64→Cys and Ser65→Ala (SEQ ID NO:5);

[0068] Phe64→Cys and Ser65→Thr;

[0069] Phe64→Leu and Ser65→Thr;

[0070] Phe64→Met and Ser65→Ala (SEQ ID NO:6);

[0071] Phe64→Met and Ser65→Thr;

[0072] Phe64→Met, Ser65→Phe and Tyr66→Phe;

[0073] Phe64→Met, Ser65→Phe and Tyr66→Lys;

[0074] Phe64→Thr and Ser65→Cys; and

[0075] Phe64→Val and Ser65→Cys

[0076] Other suitable mutations and mutant GFP amino acid sequences maybe determined by one of ordinary skill without undue experimentationaccording to the methods described herein and others that are known inthe art. As a practical matter, whether a particular mutation orcombination of mutations produces a mutant GFP that may have theabove-described desirable properties (e.g., higher expression inmammalian cells, higher fluorescence intensity under UV or white lightillumination) may be determined by one of ordinary skill using themutation, transfection, expression and detection methods described indetail below in the Examples, as well as using standard techniques thatare routine in the art.

[0077] Following mutagenesis by any of the above-described methods, theresulting nucleic acid molecules encoding the mutant GFPs may beinserted into one or more vectors, such as those described above, whichare preferably expression vectors. A particularly preferred vector forcontaining the present mutant GFP nucleic acid molecules isp-GreenLantern-2 (FIG. 6). Methods for producing the mutant GFP-vectorconstructs will be familiar to those of ordinary skill, and are providedin detail below in Example 1.

[0078] Once they have been constructed, the vectors comprising themutant GFP nucleic acid molecules may be formulated into a variety ofcompositions, such as solutions (e.g., buffer solutions) to be used intransfecting host cells. Alternatively, the vector constructs may bepurified and stored according to standard techniques for handlingrecombinant DNA plasmid vectors (Sambrook, J., et al., MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, pp. 1.3-1.20 (1989)).

[0079] More preferably, the mutant GFP-containing plasmid vectors aretransformed into a competent host cell. Any competent host cell may beused, including those of bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces spp.), insects (e.g., Spodoptera spp.) and mammals (e.g.,CHO or BHK cells), although a competent strain of E. coli such as DH10B(Invitrogen Corporation, Carlsbad, Calif.) is most preferably used.Transformation of mutagenized GFP cDNAs into host cells may beaccomplished by any technique generally used for introduction ofexogenous DNA, including the chemical, viral, electroporation,lipofection and microinjection methods that are well-known in the art.Particularly preferred methods for transformation includeelectroporation and liposome-mediated transfection (lipofection), thelatter most preferably being accomplished using LIPOFECTAMINE™(Invitrogen Corporation, Carlsbad, Calif.).

[0080] After expansion of transformed cultures, mutated GFP cDNA isisolated from the host cells by routine methods (Sambrook, J., et al.,Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press, pp. 1.21-1.52 (1989)) and issubcloned into a plasmid backbone for use in subsequent transfections.Most preferably, this plasmid backbone is the pGreenLantern-2 backbone(see FIG. 6) which contains a universal sequencing primer downstreamfrom a CMV enhancer promoter and an NsiI site immediately upstream ofthe CMV promoter allowing excision of the promoter region, along withXbaI, XhoI and HindIII sites in place of the 3′ NotI site inpGreenLantern-1 (FIG. 4).

[0081] Fusion sequences of GFP cDNA with nucleotide sequences encodingproteins of interest may be prepared by cloning the desired sequence(s)into pGreenLantern-2 at the 5′ multiple cloning site using standardtechniques. These fusion constructs allow the use of the mutant GFPs ofthe present invention as reporters of transfection efficiency. Inaddition, fusion constructs such as these will allow a directexamination of the expression, biochemistry and localization of thefused proteins intracellularly.

[0082] Alternatively, to examine the structure and function ofregulatory sequences (e.g., promoters, enhancers, inhibitors) in nativegenes, the GFP mutant cDNAs may be directly transfected or inserted,using routine methods, into target genomic or extrachromosomal DNAsequences in host cells (Chalfie, M., et al., Science 263:802-805(1994)).

[0083] Transfection of Hosts With GFP Mutants

[0084] Target cells to be transfected with cDNAs comprising mutant GFPs(either fused or unfused to accessory sequences) are grown andmaintained in culture according to routine methods. Cells may betransfected with mutant GFP cDNA by any method described above, althoughelectroporation or liposome-mediated transfection (particularly usingLIPOFECTAMINE™) are preferred. Following transfection, cells areincubated for 12-48 hours, preferably 18-24 hours and most preferablyfor about 24 hours. Transfected cells may then be examined for theexpression of mutant GFP, manifested as green intracellularfluorescence. With standard optical filters routinely used for examiningfluorescein (typically excitation wavelength of about 475 nm, dichroicfilter of 485 nm, emission wavelength of about 490 nm), thisfluorescence may be examined qualitatively, for example by fluorescencemicroscopy, or quantitatively, for example by spectrofluorimetry or flowcytofluorimetry. In addition, transfected cells expressing relativelyhigh amounts of mutant GFPs of the present invention may be separatedfrom non-transfected cells, or from those expressing lower levels ofGFP, by fluorescence-based single cell separation techniques such asfluorescence-activated cell sorting. Alternatively, transfected cellsexpressing mutant GFPs that fluoresce under white light illumination,particularly those having amino acid sequences as set forth in SEQ IDNOs: 5 and 6, may be examined by the above-described qualitative andquantitative methods using standard white light optics (e.g.,incandescent or halogen lighting, or sunlight).

[0085] These transfected host cells may also be used in methods for theproduction of mutant GFPs of the invention. Such methods may comprise,for example, culturing the above-described host cells under conditionsfavoring the production of the mutant GFPs by the host cells, andisolating the mutant GFPs from the host cells and/or the culture mediumin which the host cells are cultured. Typical host cell cultureconditions favoring production of recombinant proteins, such as thepresent mutant GFPs, are well-known in the art (see, e.g., Sambrook, J.,et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)). The mutantGFPs produced by these methods may then be isolated by any of a numberof protein purification techniques, such as chromatography (preferablyaffinity chromatography, HPLC or FPLC), salt extraction (such asammonium sulfate precipitation), electrophoresis, dialysis, or acombination thereof, to produce isolated mutant GFPs of the invention.These mutant GFPs may then be stored until use (preferably attemperatures below 0° C., more preferably at about −20° C. to about −70°C.), or they may be formulated into compositions. Preferred suchcompositions may comprise, for example, one or more of the mutant GFPsof the invention and one or more additional components, such as one ormore buffer salts, one or more inorganic salts or ions thereof, one ormore detergents, one or more preservatives, and the like, preferably inan aqueous or organic solvent.

[0086] Detection Methods

[0087] In additional embodiments, the invention relates to methods ofdetecting the presence of a mutant GFP, or of a cell (such as aprokaryotic or eukaryotic, including mammalian, cell) expressing amutant GFP. Such methods of the invention may comprise, for example,illuminating the mutant GFP or cell expressing the mutant GFP with asource of white light under conditions such that the mutant GFP or cellexpressing the mutant GFP emits visible fluorescent light. In thepresent methods, the illumination source may be any light sourceemitting white (i.e., visible) light, including but not limited to anincandescent light source, a fluorescent light source, a halogen lightsource, sunlight, and the like. When illuminated by such a white lightsource, mutant GFPs, such as those of the present invention, will emitfluorescent light of various visible wavelengths (depending upon thespecific mutations contained in the mutant GFP, as described above),which may be detected by eye or by any of the above-describedqualitative or quantitative mechanical means.

[0088] Kits

[0089] In other preferred embodiments, the compositions of the presentinvention may be assembled into kits for use in transfecting host cellswith the nucleic acid molecules encoding the present mutant GFPs, or forlabeling target polypeptides with the present mutant GFPs. Host celltransfection kits according to the present invention may comprise atleast one container containing one or more of the above-describednucleic acid molecules encoding a mutant GFP (or a compositioncomprising one or more of the nucleic acid molecules or plasmidsdescribed above), which nucleic acid molecule preferably comprisesplasmid pGreenLantern-2/A1 or plasmid pGreenLantern-2/A4 (see Example 1below). These transfection kits of the invention may optionally furthercomprise at least one additional container which may contain, forexample, a reagent for delivering the mutant GFP nucleic acid moleculeinto a host cell; in preferred kits, this reagent may comprise aliposome and most preferably LIPOFECTAMINE™. Polypeptide labeling kitsaccording to the present invention may comprise at least one containercontaining, for example, a mutant GFP such as those described above (ora composition of the invention comprising a mutant GFP), which ispreferably a mutant GFP having an amino acid sequence as set forth inSEQ ID NO:5 or SEQ ID NO:6. These labeling kits of the invention mayoptionally further comprise at least one additional container which maycontain, for example, a reagent for covalently linking the mutant GFP tothe target polypeptide.

[0090] Use of Mutant GFPs

[0091] The mutant GFPs and kits of the present invention may be used ina variety of applications. For example, the mutant GFP cDNAs are usefulas reporter genes that allow a determination of transfection efficiencyand success (Chalfie, M., et al., Science 263:802-805 (1994)).Alternatively, the mutant proteins themselves may be used as fluorescentlabels suitable for detectably labeling other proteins, nucleic acids orparticulates to be used in a variety of applications (Heim, R., et al.,Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Yokoe, H., and Meyer,T., Nature Biotech. 14:1252-1256 (1996)), such as labeling antibodiesused in infectious disease diagnostic methods; mutant GFPs may beattached to target polypeptides and proteins by a variety of methodsthat are well-known to one of ordinary skill in the art, including theuse of chemical coupling reagents. In addition, fusion complexes betweenGFP and other proteins may be constructed to allow closer and moresensitive determinations of the expression, biochemistry, localizationand trafficking of intracellular proteins in many host cells (Heim, R.,et al., Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Wang, S., andTulle, H., Nature 369:400-403 (1994); Marshall, J., et al., Neuron14:211-215 (1995); Rizzuto, R., et al., Curr. Biol. 6:183-188 (1996)).Importantly, use of the mutant GFPs that emit fluorescence whenilluminated by white light will spare the user considerable expense andtechnical difficulty that can accompany the use of fluorescent opticsfor the examination of fluorescent reporter genes such as GFP.

[0092] It will be readily apparent to one of ordinary skill in therelevant arts that other suitable modifications and adaptations to themethods and applications described herein are obvious and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Construction of Mutant GFP cDNAs

[0093] Plasmids. As depicted in FIG. 5, pGreenLantern-1 (InvitrogenCorporation, Carlsbad, Calif.; catalogue no. 10642) contains thehumanized S65T mutant GFP cDNA (FIG. 1; SEQ ID NOs:1, 2) (Evans, K., etal., FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol.70:4646-4654 (1996)). This plasmid serves as the source of the GFP DNAsequence used for mutagenesis. As depicted in FIG. 6, pGreenLantern-2contains a universal sequencing primer downstream of the CMV promoteralong with an NsiI site immediately upstream of the CMV promoterallowing excision of the promoter region. It also contains XbaI, XhoIand HindIII sites in place of the 3′ NotI site in pGreenLantern-1. Astop codon in the 5′ multiple cloning site of pGreenLantern-1 wasshifted out of frame to allow possible fusions to GFP inpGreenLantern-2.

[0094] Mutations to GFP cDNA by UDG cloning. PCR was performed in an MJResearch DNA Engine™ thermal cycler using the following conditions: 94°C. for 60 seconds, 94° C. for 30 seconds, 55° C. for 30 seconds and 72°C. for 4 minutes, repeated for 20 cycles. Sense oligonucleotide primerscontaining specific mismatches to the wt-GFP sequence (SEQ ID NOs:7-15;Table 2) were obtained from Invitrogen Corporation (Carlsbad, Calif.).TABLE 2 Sense Oligonucleotides Used for UDG Cloning Mutations. AminoSingle-stranded Acid Oligonucleotide SEQ Muta- Sequence ID Vector tions(5′ to 3′) NO: pGreenLantern-2/A1 Cys64, CAACACUGGUCACUACCTG-  7 Ala65CGCCTATGGCGTGC pGreenLantern-2/A2 Cys64, CCAACACUGGUCACUACCT-  8 Thr65GCACCTATGG pGreenLantern-2/A3 Leu64, CAACACUGGUCACUACCCT-  9 Thr65CACCTATGGCGTGCAGT pGreenLantern-2/A4 Met64, CAACACUGGUCACUACAAT- 10Ala65 GGCCTATGGCGTGCAGTGCT pGreenLantern-2/A5 Met64,CAACACUGGUCACUACCAT- 11 Thr65 GACCTATGGCGTGCAGTGCT pGreenLantern-2/A6Met64, CAACAGUGGUCACUACCAT- 12 Phe65, GTTCTTCGGCGTGCAGTGCT Phe66pGreenLantern-2/A7 Met64, CAACACUGGUCACUACGAT- 13 Phe65,GTTCAAGGGCGTGCAGTGCT Lys66 pGreenLantern-2/A8 Thr64,CAACACUGGUCACUACCAC- 14 Gys65 ATGCTATGGCGTGCAGT pGreenLantern-2/A9Val64, CAACACUGGUCACUACCGT- 15 Cys65 GTGCTATGGCGTGCAGT

[0095] The antisense oligonucleotide primer used for each mutation sethad the following sequence: 5′-AGU-GAC-CAG-UGU-UGG-CCA-AGG-CAC-AGG-GAG-CTT-3′ (SEQ ID NO:16). Thetemplate plasmid used was pGreenLantern-1 (FIG. 5) with a universalreverse sequencing primer incorporated into the backbone. Amplificationsreactions contained 1×PCR buffer, 10 micromolar deoxynucleosidetriphosphates, 25 picomoles of each primer (sense and antisense) and 10nanograms of template DNA in a 50 microliter volume. After 6, 9 and 20PCR cycles were completed, 10 microliter samples were taken and checkedvia agarose gel electrophoresis for excess background. Two 20 microlitersamples of each 6-cycle aliquot were digested with DpnI at 37° C. for 30minutes, then at 75° C. for 15 minutes and allowed to cool to roomtemperature. One of the samples from each reaction (four samples in all)was treated with one unit of uracil DNA glycosylase (UDG) at 37° C. for30 minutes (Nisson, P., et al., PCR Meth. Appl. 1:120-123 (1991)). PCRsamples were then transformed into 100 microliters of MAX EfficiencyDH10B™ Competent Cells (Invitrogen Corporation; Carlsbad, Calif.). Themutated portion of the GFP cDNA was then subcloned with a NotI and BamHIdigest into the pGreenLantern-2 backbone (FIG. 6) which was notsubjected to PCR (Sambrook, J., et al., Molecular Cloning, a LaboratoryManual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress (1989)). This approach yielded nine separate mutant GFP plasmidvectors, designated pGreenLantern-2/A1 through pGreenLantern-2/A9 (Table2), each with a specific mutation or set of mutations within the GFPchromophore region at amino acids 64-66.

Example 2 Growth and Transfection of Host Cells With Mutant GFPs

[0096] Cell Culture. Chinese hamster ovary cells (CHO-K1, obtained fromAmerican Type Culture Collection (ATCC), Rockville, Md.) were culturedin D-MEM (4,500 milligrams/liter D-glucose with L-glutamine and phenolred) plus 10% fetal bovine serum (FBS), 0.1 millimolar nonessentialamino acids, 2.5 units per milliliter penicillin and 2.5 micrograms permilliliter streptomycin (Freshney, R. I., Culture of Animal Cells: AManual of Basic Techniques, 3rd Ed., New York: Wiley-Liss (1994)). Cellswere grown at 37° C. in a 5% CO₂/air incubator. All media and reagentswere from Invitrogen Corporation, Carlsbad, Calif.

[0097] Transfection. CHO-K1 cells were plated at 2×10⁵ cells per wellinto six-well (35 millimeter diameter) plates one day prior totransfection. Immediately before transfection, cells were rinsed withmedium containing no serum or antibiotics. LIPOFECTAMINE™ reagent wasdiluted into 100 microliters of OPTI-MEM-I Reduced Serum Medium (withoutFBS) to give a final concentration of LIPOFECTAMINE of 6 microliters perwell. DNA was diluted separately to a concentration of 1 microgram perwell in 100 microliters of OPTI-MEM-I. Transfection complexes wereformed by combining diluted lipid and DNA and incubating for 30 minutesprior to addition to cells. Transfection complexes were then diluted 1:5with D-MEM containing no FBS or antibiotics and added to the rinsedcells. Cells were transfected for five hours at 37° C., then fed with anequal volume of D-MEM containing 20% FBS, 0.1 millimolar nonessentialamino acids, and no antibiotics. Cells were grown overnight at 37° C.,5% CO₂/air. In some studies, cells were grown for 48 hours; in thesestudies, transfection complexes were removed from cells 24 hours afteraddition and cells were fed with 2 milliliters per well of completemedium.

[0098] Regardless of the vector used, host cells transfected with themutant GFP genes demonstrated approximately equivalent growth rates ascontrol cells transfected with the wildtype GFP gene or with otherreporter genes (e.g., β-gal). These results indicate that transfectionwith the mutant GFP cDNAs of the present invention does not adverselyaffect the growth or culturability of the host cells more thantransfection with any other reporter vector.

Example 3 Characterization of GFP Mutants Expressed in Eukaryotic Cells

[0099] Formalin Fixation. Transfected host cells were rinsed inDulbecco's Phosphate Buffered Saline (PBS), then fixed in a solution of10% formalin in PBS for one hour. Formalin was then removed, and cellswere rinsed and stored in PBS at 4° C. until being analyzed.

[0100] Fluorescence Microscopy. Formalin-fixed cells were examined andphotographed using an inverted phase contrast fluorescence microscopeequipped with FITC filters (excitation 475 nm/dichroic 485 nm/barrier490 nm) and a 50 watt mercury arc bulb at 1.25 volts. A 40×-poweradjustable non-phase objective was used for all micrographs, which weretaken through blue, neutral and FITC filters using Kodak Ektachrome ASA400 Daylight (for slides) or Kodak Gold ASA 400 Daylight (for prints).All exposures were for 12 seconds to allow unbiased comparison offluorescence intensity.

[0101] Flow Cytofluorimetry. Flow cytofluorimetry was performed ontransfected CHO-K1 cells that were trypsinized and suspended in PBS plus10% formalin at a concentration of less than 10⁶ cells per milliliter.Measurements were made on a Coulter EPICS® XL-MCL flow cytometer using a15 megawatt argon ion laser. Filters used were 488 nm excitation, 500 nmdichroic LP/525 nm band pass for FL1 (green channel) and 575 bandpass/600 nm dichroic LP for FL2 (orange channel). Samples consisted of20,000 events using PMT voltages of 100 volts for side scatter andforward scatter, 496 volts for FL1 and 505 volts for FL2, all withintegral gain set to 1.0. Color compensation included 7.9% orange signalin FL1 and 3.2% green signal in FL2.

[0102] Results. As shown in Table 3, the GFP mutants of the presentinvention displayed varying intensities and kinetics of formation intransfected cells. Two of these mutants, designated “A1” (phenylalaninemutated to cysteine at position 64; serine mutated to alanine atposition 65; FIG. 3; SEQ ID NO:5) and “A4” (phenylalanine mutated tomethionine at position 64; serine mutated to alanine at position 65;FIG. 4; SEQ ID NO:6) were exceptionally bright. As shown in FIGS. 7-9,CHO cells transfected with plasmid pGreenLantern-2/A1 (FIG. 7) or withplasmid pGreenLantern-2/A4 (FIG. 8) demonstrated a dramatic increase ingreen fluorescence intensity over cells transfected with the humanizedS65T mutation of pGreenLantern-1 (FIG. 9) when viewed at 24 hourspost-transfection using FITC optics. TABLE 3 Effects of Point Mutationson GFP Fluorescence Intensity. Vector Amino Acids Fluorescence ResultsWildtype GFP Phe64, Ser65 λ_(ex) = 395 nm (major), 470 nm (minor); 48hours required for detection S65T Phe64, Thr65 6-fold increase inintensity over wildtype pGreenLantern-1 Phe64, Thr65 22-fold increase inintensity (humanized) over wildtype pGreenLantern- Cys64, Ala65 6-foldincrease in intensity over S65T 2/A1 pGreenLantern- Cys64, Thr65 22-foldincrease in intensity 2/A2 over wildtype pGreenLantern- Leu64, Thr656-fold increase in intensity over S65T 2/A3 pGreenLantern- Met64, Ala656-fold increase in intensity over S65T 2/A4 pGreenLantern- Met64, Thr65Slight increase in intensity over 2/A5 pGreenLantern-1 pGreenLantern-Met64, Equivalent to wildtype 2/A6 Phe65, Phe66 pGreenLantern- Met64,Equivalent to wildtype 2/A7 Phe65, Lys66 pGreenLantern- Thr64, Cys65Equivalent to wildtype 2/A8 pGreenLantern- Val64, Cys65 Slight increasein intensity over 2/A9 pGreenLantern-1

[0103] Other mutants produced in the present studies were lesssatisfactory (Table 3). For example, mutants A5 (phenylalanine mutatedto methionine at position 64; serine mutated to threonine at position65) and A9 (phenylalanine mutated to valine at position 64; serinemutated to cysteine at position 65) gave only slightly betterfluorescence than the humanized S65T mutation of pGreenLantern-1. It ispossible that the highly reactive cysteine at position 65 in mutant A9may interfere with the formnation of the three amino acid heterocyclicring required for GFP fluorescence (Cody, C. W., Biochemistry32:1212-1218 (1993)).

[0104] Mutant A2 (phenylalanine mutated to cysteine at position 64;serine mutated to threonine at position 65) was equal in fluorescence tothe humanized S65T pGreenLantern-1 (Evans, K., et al., FOCUS 18(2):40-43(1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654 (1996)), whilemutants A6 (phenylalanine mutated to methionine at position 64; serinemutated to phenylalanine at position 65; tyrosine mutated tophenylalanine at position 66), A7 (phenylalanine mutated to methionineat position 64; serine mutated to phenylalanine at position 65; tyrosinemutated to lysine at position 66) and A8 (phenylalanine mutated tothreonine at position 64; serine mutated to cysteine at position 65)demonstrated a decreased fluorescence intensity and were, in fact,equivalent to wt-GFP. No shift in excitation or emission spectra wasdetected with these three mutants, however, as no fluorescence wasobserved using ultraviolet or rhodamine filter combinations.

[0105] These results were also observed via flow cytometry. As shown inFIG. 10, CHO-K1 cells transfected with the A1 and A4 mutant GFPsdemonstrated a dramatic increase in fluorescence over wildtype and A6-A8mutants within 24 hours of transfection. This high level of fluorescencewas maintained, particularly for cells transfected with the A4 mutantGFP, for at least 48 hours after transfection (FIG. 11).

[0106] Mutations at certain amino acid positions outside the chromophorewere also examined for their effects on GFP fluorescence. Mutation ofGln69→Asn in the A4 mutant resulted in a dramatic decrease influorescence relative to the A4 mutant itself, as did mutation ofVal163→Ala and Ile167→Thr in the A4 mutant.

[0107] Together, these results indicate that the most preferablemutations for providing highly fluorescent, rapidly expressed GFPs arethose in which only one reactive amino acid is present at eitherposition 64 or 65, as in the A1 (Phe64→Cys; Ser65→Ala; SEQ ID NO:5) andA4 (Phe64→Met; Ser65→Ala; SEQ ID NO:6) mutants.

Example 4 Characterization of GFP Mutants Expressed in Prokaryotic Cells

[0108] To examine the efficacy of expressing mutant GFPs in prokaryoticcells, mutant GFP cDNAs were subcloned into the bacterial pProEX HTbvector (FIG. 12). GFP cDNA was excised by NotI and XbaI digestion frompGreenLantern-2 (FIG. 6) containing the mutations at positions 64, 65and/or 66 (mutants A1 through A9) shown in Table 3. The bacterial vectorpProEX HTb (FIG. 12) was also digested with the same enzymes. The pProEXHTb backbone and GFP fragments were ligated, to form the correspondingtransfection vectors containing the respective mutant GFP fragments:pProEXA1, pProEXA2, pProEXA3, pProEXA4, pProEXA5, pProEXA6, pProEXA7,pProEXA8 and pProEXA9. These vectors were then individually transformedinto 100 μl of DH10B E. coli host cells; control cells were alsoprepared that had been transfected with a construct containing the S65Tmutant described in Examples 1-3 above. Cells were plated ontoampicillin/IPTG plates and incubated overnight at 37° C., and colonieswere then picked and screened for fluorescence under long ultraviolet(UV) or blue illumination.

[0109] Colonies containing the A1, A2, A3, A4, A5, A9 and S65T mutantGFPs all demonstrated green fluorescence when illuminated with long UVor blue light, while those containing the A6, A7 and A8 mutant GFPsdemonstrated no fluorescence under these conditions. These results areconsistent with those observed in eukaryotic cells, as shown in Example3 above, and indicate that mutant GFPs may be successfully transfectedinto and expressed in prokaryotic cells.

Example 5 Visible Light Excitation of GFP Mutants

[0110] To examine the ability of mutant GFPs to emit fluorescence whenilluminated by white light, E. coli cells were transfected and plated asdescribed above in Example 4. Colonies were then picked and examined forfluorescence upon illumination by incandescent light, fluorescent indoorlighting, or sunlight.

[0111] Upon induction of the host cells with IPTG, cells transformedwith the vector comprising the A4 GFP mutation unexpectedly exhibitedbright green light emission under normal daylight conditions, withoutthe need for excitation with UV light. Similar results were observed forcells transformed with the A3 mutant GFP. Cells containing the A1 and A5mutant GFPs were also seen to be less (but still observably) fluorescentunder white light illumination. Conversely, only very weak emission oflight was observed under white light illumination in the cellstransformed with the vectors comprising only the S65T, A2 and A9mutations. Cells comprising the A6, A7 and A8 mutations exhibited nofluorescence when illuminated by white light.

[0112] When plates containing these mutants were stored in the dark at4° C. for 38 days, however, all of the colonies except those containingthe A6, A7 or A8 mutant GFPs were seen to be more intensely fluorescentunder white light illumination. Colonies containing the A3, A4 and A5mutants were more fluorescent under these conditions than were thosecontaining the A1, A2, A9 and S65T mutants, although all coloniesfluoresced more brightly than they did in freshly plated cells (i.e.,when observed within 24-48 hours of transfection). When these plateswere allowed to warm to room temperature, the fluorescence in coloniescontaining the A1, A2, A9 and S65T mutants decreased, while that incolonies containing the A3, A4 and A5 mutants remained brightlyfluorescent.

[0113] It is possible that the increased fluorescence observed in storedplates may have been due to accumulation of mutant protein in the cellsover time in storage, indicating a dependence of white lightfluorescence upon intracellular concentration of the GFP. To test thisnotion, a 6His-tagged A4 GFP construct prepared and isolated by metalaffinity chromatography according to standard techniques (see Ausubel,F. M., et al., in Current Protocols in Molecular Biology, New York: JohnWiley & Sons, Inc., pp. 10.11.10-10.11.24 (1996)), was examined forfluorescence under blue, red and white light at various proteinconcentrations in solution. At a concentration of about 1.5 μg/ml, thepurified A4 GFP was brightly fluorescent under sunlight and fluorescentindoor white lighting, as well as under blue light; no fluorescence wasobserved, however, under red light. This highly concentrated A4 GFPsolution became nonfluorescent upon boiling, but was at least slightlyfluorescent up to a temperature of about 82° C. When diluted to 0.1μg/ml, however, the A4 GFP solution fluoresced brightly under blue light(closer in wavelength to the excitation maximum of GFP which is in theUV range), but did not fluoresce under white light illumination. Theseresults suggest that the increased fluorescence observed upon whitelight illumination of colonies stored for extended periods of time maybe due to accumulation of GFP protein in the cells.

[0114] Taken together, these results indicate that prokaryotic cellscontaining the A3 or A4 mutant GFPs, and to a lesser extent the A1 andA5 mutant GFPs, can emit light without the addition of an exogenoussubstrate or the use of ultraviolet irradiation. Use of these GFPconstructs thus provides advantages over other visible light reportervectors which require the use of exogenous substrates, and over otherfluorescent reporter vectors which require UV irradiation which mayinduce undesirable mutations in the host cells.

Example 6 Additional GFP Mutations

[0115] To examine the effects of alternative point mutations on GFPfluorescence, mutations are targeted at the tryptophan residue atposition 67 (the only tryptophan residue in the entire GFP moleculewhich is located in the unique motif Pro-Val-Pro-Trp-Pro (SEQ IDNO:17)). To accomplish this mutation, oligonucleotides are designed tomutate Trp57→His or Trp57→Tyr, in conjunction with the Ser65→Thr mutant(SEQ ID NO:2) or the Phe64→Met; Ser65→Ala mutant (SEQ ID NO:6). Thesemutants are made in the bacterial vector pProEX HTb as described inExample 4, using specific oligonucleotides designed to provide thedesired mutations. The vector constructs are then transfected into hostcells and characterized as above for their fluorescence.

[0116] In a similar fashion, mutations are made at other amino acidpositions outside of the GFP chromophore region. For example, mutationsare made at Arg96, which is probably responsible for stabilizingresonance structures of the imidazolidone 5-membered ring during ringformation and possibly during excitation, and is therefore a target formore rapid ring formation and, hence, faster detection of fluorescence.Mutations involving this residue include Arg96→His.

[0117] Mutations are also possible at Phe46, which along with Phe64separates the 5-membered chromophore ring from direct contact with thesingle tryptophan in the Ser65→Thr GFP (SEQ ID NO:2). By allowing directhydrogen bonding between Trp57 and the ring structure, efficient energytransfer is possible as with the Phe64→Leu; Ser64→Thr mutant. Mutationsinvolving this residue include Phe46→Leu or other hydrophobic residuesthat promote hydrogen bonding.

[0118] Mutations are also made at Leu221 and Phe223, which are involvedin dimer formation. Only three hydrophobic residues are in the dimercontact region; all others are hydrophobic. By mutating Leu221 and/orPhe223 to a hydrophilic or “neutral” residue such as glycine, GFPaggregation, which can be a problem with GFP fusion constructs, may beinhibited.

[0119] Mutations are also made at His148, which probably stabilizes thefluorophore and forms hydrogen bonds with Tyr66 and Gln94. Mutations ofHis148 to a residue with a different charge or a different pKa are madeto allow alteration of the excitation and emission spectra of GFP,similar to results seen with Tyr66→His which results in bluefluorescence by GFP.

[0120] Finally, mutations introducing a second 5-membered ring structureinto the α-helix of GFP are made, to allow increased fluorescenceintensity of the resultant GFP.

[0121] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0122] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

1 17 1 717 DNA Aequorea victoria, gfp(h) S65T mutant cDNA clone 1 atgagc aag ggc gag gaa ctg ttc act ggc gtg gtc cca att ctc gtg 48 Met SerLys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaactg gat ggc gat gtg aat ggg cac aaa ttt tct gtc agc gga gag 96 Glu LeuAsp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaaggt gat gcc aca tac gga aag ctc acc ctg aaa ttc atc tgc 144 Gly Glu GlyAsp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 acc act ggaaag ctc cct gtg cca tgg cca aca ctg gtc act acc ttc 192 Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 acc tat ggc gtgcag tgc ttt tcc aga tac cca gac cat atg aag cag 240 Thr Tyr Gly Val GlnCys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttcaag agc gcc atg ccc gag ggc tat gtg cag gag aga 288 His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 acc atc ttt ttc aaagat gac ggg aac tac aag acc cgc gct gaa gtc 336 Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttc gaa ggt gacacc ctg gtg aat aga atc gag ttg aag ggc att 384 Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gac ttt aag gaa gatgga aac att ctc ggc cac aag ctg gaa tac aac 432 Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tcc cac aatgtg tac atc atg gcc gac aag caa aag aat ggc 480 Tyr Asn Ser His Asn ValTyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aag gtc aacttc aag atc aga cac aac att gag gat gga tcc gtg 528 Ile Lys Val Asn PheLys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 cag ctg gcc gaccat tat caa cag aac act cca atc ggc gac ggc cct 576 Gln Leu Ala Asp HisTyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtg ctc ctc ccagac aac cat tac ctg tcc acc cag tct gcc ctg tct 624 Val Leu Leu Pro AspAsn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aacgaa aag aga gac cac atg gtc ctg ctg gag ttt gtg 672 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 acc gct gct gggatc aca cat ggc atg gac gag ctg tac aag 714 Thr Ala Ala Gly Ile Thr HisGly Met Asp Glu Leu Tyr Lys 225 230 235 tga 717 2 238 PRT Aequoreavictoria, gfp(h) S65T mutant cDNA clone 2 Met Ser Lys Gly Glu Glu LeuPhe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Thr Tyr Gly Val Gln CysPhe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe LysAsp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu GlyAsp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe LysGlu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr AsnSer His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu PheVal 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225 230 235 3 717 DNA Aequorea victoria, gfp10 cDNA clone 3 atg agt aaagga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gatggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp GlyAsp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gatgca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act act gga aaa ctacct gtt cca tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu ProVal Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgcttt tca aga tac cca gat cat atg aaa cag 240 Ser Tyr Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag agtgcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttc aaa gat gacggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc cttgtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu ValAsn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aacatt ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn IleLeu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cac aat gta tacatc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His Asn Val Tyr IleMet Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaaatt aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys IleArg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tatcaa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aaccat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn HisTyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aagaga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys ArgAsp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg att acacat ggc atg gat gaa cta tac aaa 714 Thr Ala Ala Gly Ile Thr His Gly MetAsp Glu Leu Tyr Lys 225 230 235 taa 717 4 238 PRT Aequorea victoria,gfp10 cDNA clone 4 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val ProIle Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe SerVal Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr LeuLys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr LeuVal Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro AspHis Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu GlyTyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr LysThr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn ArgIle Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr IleMet Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe LysIle Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp HisTyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp ProAsn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr AlaAla Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 5 238 PRTAequorea victoria, A1 mutant 5 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly HisLys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu Val Thr Thr Cys 50 55 60 Ala Tyr Gly Val Gln Cys Phe Ser ArgTyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr LeuVal Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys ValAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln LeuAla Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 ValLeu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 2356 238 PRT Aequorea victoria, A4 mutant 6 Met Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val AsnGly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr Met 50 55 60 Ala Tyr Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys SerAla Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn SerHis Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 IleLys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225230 235 7 33 DNA Artificial sequence Synthetic oligonucleotide 7caacacuggu cacuacctgc gcctatggcg tgc 33 8 29 DNA Artificial sequenceSynthetic oligonucleotide 8 ccaacacugg ucacuacctg cacctatgg 29 9 36 DNAArtificial sequence Synthetic oligonucleotide 9 caacacuggu cacuaccctcacctatggcg tgcagt 36 10 39 DNA Artificial sequence Syntheticoligonucleotide 10 caacacuggu cacuacaatg gcctatggcg tgcagtgct 39 11 39DNA Artificial sequence Synthetic oligonucleotide 11 caacacuggucacuaccatg acctatggcg tgcagtgct 39 12 39 DNA Artificial sequenceSynthetic oligonucleotide 12 caacacuggu cacuaccatg ttcttcggcg tgcagtgct39 13 39 DNA Artificial sequence Synthetic oligonucleotide 13 caacacuggucacuaccatg ttcaagggcg tgcagtgct 39 14 36 DNA Artificial sequenceSynthetic oligonucleotide 14 caacacuggu cacuaccaca tgctatggcg tgcagt 3615 36 DNA Artificial sequence Synthetic oligonucleotide 15 caacacuggucacuaccgtg tgctatggcg tgcagt 36 16 33 DNA Artificial sequence Syntheticoligonucleotide 16 agugaccagu guuggccaag gcacagggag ctt 33 17 5 PRTAequorea victoria 17 Pro Val Pro Trp Pro 1 5

What is claimed is:
 1. A nucleic acid molecule encoding a mutant GreenFluorescent Protein, said mutant Green Fluorescent Protein having anamino acid sequence comprising an amino acid residue lacking an aromaticring structure at position 64 and an amino acid residue having a sidechain no longer than two carbon units in length at position 65, with theprovisos that if said residue at position 64 is leucine then saidresidue at position 65 is not cysteine or threonine; if said residue atposition 64 is valine then said residue at position 65 is not alanine;if said residue at position 64 is methionine then said residue atposition 65 is not glycine; and if said residue at position 64 isglycine then said residue at position 65 is not cysteine.